Antibody molecules (also known as immunoglobulins) have a twofold symmetry and are composed of two identical heavy chains and two identical light chains, each containing variable and constant domains. The variable domains of the heavy and light chains combine to form an antigen-binding site, so that both chains contribute to the antigen-binding specificity of the antibody molecule. The basic tetrameric structure of antibodies comprises two heavy chains covalently linked by a disulphide bond. Each heavy chain is in turn attached to a light chain, again via a disulphide bond, to produce a substantially “Y”-shaped molecule.
There are two types of light chain: Lambda (λ) and Kappa (κ). There are approximately twice as many κ as λ molecules produced in humans, but this is quite different in some mammals. Each chain contains approximately 220 amino acids in a single polypeptide chain that is folded into one constant and one variable domain. Plasma cells produce one of the five heavy chain types together with either κ or λ molecules. There is normally approximately 40% excess free light chain production over heavy chain synthesis. Where the light chain molecules are not bound to heavy chain molecules, they are known as “free light chain molecules” (FLCs). The κ light chains are usually found as monomers. The λ light chains tend to form dimers.
There are a number of proliferative diseases associated with antibody producing cells. FIG. 1 shows the development of B-cell lineage and associated diseases. These diseases are known as malignant B-cell diseases. They are summarised in detail in the book “Serum-free Light Chain Analysis” A. R. Bradwell, available from The Binding Site Limited, Birmingham, UK (ISBN: 07044 24894), Third Edition 2005, and the Second Edition of the book (2004, ISBN 07044 24541).
In many such diseases a plasma cell proliferates to form a monoclonal tumour of identical plasma cells. This results in production of large amounts of identical immunoglobulins and is known as monoclonal gammopathy.
Diseases such as myeloma and primary systemic amyloidosis (AL amyloidosis) account for approximately 1.5% and 0.3% respectively of cancer deaths in the United Kingdom. Multiple myeloma (MM) is the second-most common form of haematological malignancy after non-Hodgkin lymphoma. In Caucasian populations the incidence is approximately 40 per million per year. Conventionally, the diagnosis of MM is based on the presence of excess monoclonal plasma cells in the bone marrow, monoclonal immunoglobulins in the serum or urine and related organ or tissue impairment such as hypercalcaemia, renal insufficiency, anaemia or bone lesions. Normal plasma cell content of the bone marrow is about 1%, while in MM the content is typically greater than 30%, but may be over 90%.
AL amyloidosis is a protein conformation disorder characterised by the accumulation of monoclonal free light chain fragments as amyloid deposits. Typically, these patients present with heart or renal failure but peripheral nerves and other organs may also be involved.
The Binding Site Ltd have previously developed a sensitive assay that can detect the free κ light chains and, separately, the free λ light chains (PCT/GB2006/000267, published as WO2006/079816). This method uses a polyclonal antibody directed towards either the free κ or the free λ light chains. The detection of free light chains (FLC) is discussed in detail in the book by A. R. Bradwell. The possibility of raising such antibodies was also discussed as one of a number of different possible specificities, in WO97/17372. This form of assay has been found to successfully detect free light chain concentrations. Furthermore, the sensitivity of the technique is very high.
Bradwell A. R., et al. (Clin. & Applied Immunol. Reviews 3 (2002), 17-33) reviews serum free light chain immunoassays and their applications. Historically, urine concentrations of FLC have not been considered to accurately reflect plasma cell synthesis. Hence, there has been a move away from testing urine concentrations to serum-based FLC assays, using techniques such as nephelometry and immunofixation electrophoresis. The paper summarises the understanding in the art of FLC synthesis and metabolism with respect to renal function.
Approximately 12-20% of MM patients first present in acute renal failure. 10% are dialysis dependent in the long term.
Free κ and free λ are cleared by filtration through the kidneys and the rate depends on their molecular size. Monomeric free light chains, characteristically K, are cleared in 2-4 hours at 40% of the glomerular filtration rate. Dimeric free light chains, typically λ, are cleared in 3-6 hours at 20% of the glomerular filtration rate, while large molecules are cleared more slowly. Removal may be prolonged to 2-3 days in MM patients in renal failure, when serum free light chains (sFLCs) are removed by the liver and other tissues (Russo et al. (2002) Am. J. Kidney Dis. 39 899-919). In contrast, IgG has a half-life of 21 days that is not affected by renal impairment.
There are approximately 0.5 million nephrons in each human kidney. Each nephron contains a glomerulus with pores that allow filtration of serum molecules into its proximal tubule. The pore sizes are variable with a restriction in filtration commencing at about 40 kDa and being almost complete by 65 kDa. Protein molecules that pass the glomerular pores are then either absorbed unchanged or degraded in the proximal tubular cells and excreted as fragments. This is an essential mechanism to prevent loss of proteins and peptides into the urine and is very efficient. The exact pathway of free light chain is unknown but between 10-30 g per day can be processed by the kidneys, so, under normal conditions, very little free light chain passes beyond the proximal tubules.
After filtration by the glomeruli, FLCs enter the proximal tubules and bind to brush-border membranes via low-affinity, high-capacity receptors called cubulins (gp280) (Winearls (2003) “Myeloma Kidney”—Ch. 17 Comprehensive Clinical Nephrology, 2nd Ed. Eds Johnson & Feehally; Pub: Mosby). Binding provokes internalisation of the FLCs and subsequent metabolism. The concentration of the FLCs leaving the proximal tubules, therefore, depends upon the amounts in the glomerular filtrate, competition for binding uptake from other proteins and the absorptive capacity of the tubular cells. A reduction in the glomerular filtration rate increases serum FLC concentrations so that more is filtered by the remaining functioning nephrons. Subsequently, and with increasing renal failure, hyperfiltering glomeruli leak albumin and other proteins which compete with FLCs for absorption, thereby causing more to enter the distal tubules.
FLCs entering the distal tubule normally bind to uromucoid (Tamm-Horsfall protein). This is the dominant protein in normal urine and is though to be important in preventing ascending urinary infections. It is a glycoprotein (85 kDa) that aggregates into high molecular weight polymers of 20-30 units. Interestingly, it contains a short peptide motif that has a high affinity for FLCs (Ying & Sanders (2001) Am. J. Path. 158 1859-1866). Together, the two proteins form waxy casts that are more characteristically found in acute renal failure associated with light chain MM (LCMM) (see e.g. Winearls (1995) Kidney Int. 48 1347-1361). The casts obstruct tubular fluid flow, leading to disruption of the basement membrane and interstitial damage. Rising concentrations of sFLCs are filtered by the remaining functioning nephrons leading to a vicious cycle of accelerating renal damage with further increases in sFLCs. This may explain why MM patients, without apparent pre-existing renal impairment, suddenly develop renal injury and renal failure. The process is aggravated by other factors such as dehydration, diuretics, hypercalcaemia, infections and nephrotoxic drugs.
Serum FLC concentrations are abnormal in >95% of patients with MM and have a wide range of concentrations, but their inherent toxicity also varies considerably, as was shown by Sanders & Brooker using isolated rat nephrons (Sanders & Brooker (1992) J. Clin. Invest. 89 630-639). The toxicity is in part related to binding with Tamm-Horsfall protein (see e.g. Winearls (1995) Kidney Int. 48 1347-1361).
In spite of much effort to show otherwise, particular molecular charge and/or κ or λ type are not now considered relevant to FLC toxicity. Furthermore, highly polymerised FLCs (a frequent finding in MM) are probably not nephrotoxic because they cannot readily pass through the glomeruli. This may partly account for the lack of renal damage in some patients who have very high sFLC concentrations.
The amount of sFLCs necessary to cause renal impairment was recently studied by Nowrousain et al. (Clin. Cancer Res. (2005) 11 8706-8714), who showed that the median serum concentrations associated with overflow proteinuria (and hence potential for tubular damage) was 113 mg/L for κ and 278 mg/L for λ. These concentrations are approximately 5-to 10-fold above the normal serum concentrations and presumably relate to the maximum tubular reabsorption capacity of the proximal tubules. Since the normal daily production of FLC is ˜500 mg, increases in ˜5 g/day are likely to be nephrotoxic in many patients.
There have been several urine studies that have related urine FLC excretion rates to renal impairment. Typically, the associated renal impairment rises with increasing urine FLCs. One study showed that 5%, 17% and 39% of patients had renal impairment with excretion rates of <0.005 g/day, 0.005-2 g/day and >2 g/day, respectively (Blade (2003) “Management of Renal, Hematologic and Infectious Complications” in: Myeloma: Biology and Management, 3rd Ed. Eds Malpas et al.; Pub: Saunders). However, FLC excretion is an indicator of renal damage in addition to its cause.
The pre-renal load of FLCs is an important factor in renal toxicity. In an attempt to minimise renal damage, plasma exchange (PE) has been used to reduce the pre-renal load of serum free light chains. Zuchelli et al. (Kidney Int. (1988) 33 1175-1180) compared MM patients on peritoneal dialysis (control group) with plasma exchange (and haemodialysis in some patients). Only 2 of 14 in the control group had improved renal function, compared with 13 of 15 in the plasma exchange arm. Survival was also improved (P<0.01).
This early success was not repeated in subsequent controlled trials. Johnson et al. (Arch Intern. Med. (1990) 150 863-869) compared 10 patients on forced diuresis with 11 who had additional plasma exchange and found no difference in outcome. Most recently, a large series was reported by Clarke et al. (Haematologica (2005) 90 (s1) 117; Kidney Int., Mar. 2006). Half of 97 patients who were on chemotherapy, haemodialysis or a combination of the two were randomly allocated to receive plasma exchange. Again, there was no statistically significant benefit from plasma exchange.
A subsequent editorial in JASN noted a number of shortcomings in plasma exchange studies, indicating that the efficiency of plasma exchange could not be judged (Ritz E., J. Am. Soc. Nephrol (2006), 17: 914-916).